It is critical to sterilize biomedical devices, xenograft and allograft materials, due to the rapid rise of antibiotic resistance in hospital-acquired infections (HAI). For one major HAI pathogen, Clostridium difficile, the use of antibiotics promotes greater infection rates and worse outcome, as the native intestinal flora is eliminated by the strong antibiotics used (Songer and Anderson, 2006). C. difficile, Staphylococcus aureus and Pseudomonas aeruginosa are responsible for many of the estimated 2 million nosocomial infections resulting in over 100,000 deaths in 2002 and with the estimate that one in twenty hospital patients will acquire an HAI (www.cdc.gov/HAI/burden.html, www.cdc.gov/HAI/pdfs/hai/infections_deaths.pdf). The estimated costs of HAI are well over $25-35 billion, with prevention costs estimated at below $7 billion (www.cdc.gov/HAI/pdfs/hai/Scott_CostPaper.pdf). With the use of any sterilization system or antibiotic, there is a need to identify the mechanism of microbicidal action of the molecule or factor involved. Lessons learned from antibiotic resistance, antibiotic promotion of C. difficile infection and biocide resistance drive the requirement for characterizing new and long-used biocide's mechanisms of kill. Understanding the critical cellular targets for biocides enables the development of innovative, safe methods to control or eliminate the incidence of HAI. [For a novel sterilization method to be accepted for medical devices for internal and external use and for pharmaceuticals, the U.S. Food and Drug Administration (FDA) now require identification of the mechanism of biocide action.] NovaSterilis, Inc., has completed two NIH Phase II grants, resulting in a validated supercritical Carbon dioxide- peracetic acid (scCO2-PAA) sterilization system on the market for soft and hard tissue and is developing sterilization of absorbable sutures. This proposal addresses the FDA requirement for characterization of the mechanism of microbial kill by PAA in scCO2. NovaSterilis' commercial Nova 2200 system monitors the conditions required for formation and maintenance of supercritical CO2, but does not currently monitor concentrations of peracetic acid (and stabilizer hydrogen peroxide) solubilized in scCO2. [The goals of the NovaSterilis Phase I program are to identify the mechanism of biocidal action of PAA and hydrogen in scCO2 on spores of the bacterium Bacillus subtilis, one of the most resistant forms of life, and to develop and test an inline meter to monitor microbicidal levels of PAA in the system.] This work will significantly advance FDA regulatory acceptance of scCO2 sterilization with PAA and hydrogen peroxide as co-sterilants for medical devices and biologicals, as well as for food and environmental systems utilizing PAA as a sterilant or co- sterilant.